Abstract The inferior colliculus (IC) is the hub of the central auditory system. Located in the midbrain, the IC receives most of the ascending output of the auditory brainstem and provides the major auditory input to the thalamocortical system. Despite this central role in auditory processing, surprisingly little is known about the identity and function of the neurons and neural circuits that make up the IC. This is largely because the diverse properties of IC neurons have made it difficult to identify neuron types using conventional anatomical and physiological approaches. This represents a critical gap in knowledge because an understanding of the key neuronal players in the IC is essential for determining how computations in IC circuits support hearing. In other brain regions, molecular genetic approaches have proven critical for identifying neuron types and probing the function of neural circuits. Following a molecular genetic approach, we have identified three Cre-driver mouse lines that use neuropeptide promoters to drive the expression of Cre recombinase in subpopulations of IC neurons: Vasoactive Intestinal Peptide (VIP)-Cre, Neuropeptide Y (NPY)-Cre, and Somatostatin (SST)-Cre. The overall objective of this proposal is to determine how these molecularly defined classes of IC neurons integrate ascending inputs and influence postsynaptic targets. To pursue this objective, we will use brain slice electrophysiology, optogenetic circuit mapping, and anatomical approaches. In Aim 1, we will determine the intrinsic physiology, morphology, axonal projections, distribution, and neurochemistry of VIP, NPY, and SST neurons. We hypothesize that VIP, NPY, and SST neurons represent separate classes of IC neurons characterized by specific physiological and anatomical properties. In Aim 2, we will use optogenetic circuit mapping to identify and assess the functional impact of ascending and commissural sources of synaptic input to VIP, NPY, and SST neurons. In addition, we will use subcellular optogenetic circuit mapping to determine the dendritic location of synaptic inputs. Our working hypothesis is that specific neuron types integrate input from multiple sources and that ascending and commissural sources of input target different regions of the dendritic tree. In Aim 3, we will determine how VIP, NPY, and SST neurons influence postsynaptic targets in the local IC, the contralateral IC, and the auditory thalamus. This aim will use a combination of optogenetic circuit mapping and paired recordings from synaptically coupled neurons. We hypothesize that local and commissural projections provide weak, modulatory synaptic input, while thalamic projections provide strong inputs that drive the activity of thalamic neurons. The expected outcome of this research is that we will determine how three molecularly defined classes of IC neurons shape the integration of auditory information within the IC and influence the output of the IC to the thalamocortical system. These results will provide a mechanistic framework for understanding how neural circuits in the IC function and will enable future investigations to determine how the same classes of IC neurons influence sound processing in vivo.